Phil. Trans. R. Soc. B (2008) 363, 3023–3036

doi:10.1098/rstb.2008.0078

Published online 25 June 2008

Review

The genic view of plant speciation: recentprogress and emerging questions

Christian Lexer1,* and Alex Widmer2

One conanimals:

*Autho

1Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK2ETH Zurich, Institute of Integrative Biology, Plant Ecological Genetics,

Universitatstrasse 16, 8092 Zurich, Switzerland

The genic view of the process of speciation is based on the notion that species isolation may beachieved by a modest number of genes. Although great strides have been made to characterize‘speciation genes’ in some groups of animals, little is known about the nature of genic barriers to geneflow in plants. We review recent progress in the characterization of genic species barriers in plantswith a focus on five ‘model’ genera: Mimulus (monkey flowers); Iris (irises); Helianthus (sunflowers);Silene (campions); and Populus (poplars, aspens, cottonwoods). The study species in all five generaare diploid in terms of meiotic behaviour, and chromosomal rearrangements are assumed to play aminor role in species isolation, with the exception of Helianthus for which data on the relative roles ofchromosomal and genic isolation factors are available. Our review identifies the following key topicsas being of special interest for future research: the role of intraspecific variation in speciation; thedetection of balancing versus directional selection in speciation genetic studies; the timing of fixationof alleles of major versus minor effects during plant speciation; the likelihood of adaptivetrait introgression; and the identification and characterization of speciation genes and speciationgene networks.

Keywords: speciation; Mimulus; Iris; Helianthus; Silene; Populus

1. INTRODUCTIONRecent years have seen great changes in our under-standing of the nature of barriers to gene flow betweendiverging populations and species (Coyne & Orr 2004).Perhaps the most consequential of these is an extensionof Ernst Mayr’s biological species concept (BSC; Mayr1942) to accommodate a ‘genic view’ of the processof speciation (Wu 2001a). Mayr’s classic BSC seesspecies as ‘.groups of actually or potentially inter-breeding natural populations which are reproductivelyisolated from other such groups.’ (Mayr 1942). Thisview of species contains the notion of ‘whole-genomeisolation’ or ‘whole-genome reproductive isolation’(Wu 2001a,b); that is, diverging populations must beprotected from gene flow throughout their genomes todeserve the biologist’s label of good species. Bycontrast, the genic view of speciation is based on thenotion that species isolation may be achieved by amodest number of genes (Wu 2001a). Alleles at suchloci will not move freely between species because theyare selected against in a ‘foreign’ genetic background(Wu 2001b). This genic view of speciation has beenmotivated mainly by the discovery of ‘speciation genes’in Drosophila (Wu 2001a; Orr et al. 2004) and holdsgreat potential to improve our understanding of speciesboundaries and divergence in many other groups

tribution of 12 to a Theme Issue ‘Speciation in plants andpattern and process’.

r for correspondence (c.lexer@kew.org).

3023

(Noor & Feder 2006). Theoretical and empirical

work predicts an important role for genic factors—as

opposed to chromosomal or genomic factors—during

speciation (Felsenstein 1981; Via 2001). ‘Porous

genomes’ are also an expected feature of adaptive

species radiations (Gavrilets & Vose 2005). Here, we

review recent studies that address the genetic archi-

tecture of genic barriers to gene flow in plants. We

do not intend to suggest a new species concept, as

plenty of these are already available in the speciation

literature (Coyne & Orr 2004). The genic view of plant

speciation represents an update to Mayr’s (1942) BSC.

By recognizing that genealogies may vary among loci in

the genome, it facilitates the study of genetic factors

such as drift, gene flow, recombination, selection and

their interactions during divergence.

Plant evolutionary genetics has long been an

important contributor to evolutionary theory and

thinking; for example, the modern evolutionary

synthesis would not have been possible without

Gregor Mendel’s experimental work on plants. In the

twenty-first century, plants have lost nothing of their

attractiveness for evolutionary biologists interested

in testing theoretical predictions: plants can often be

crossed rather easily, and their sessile nature facili-

tates the estimation of fitness effects of individual traits,

chromosomal segments or even individual genes in

the wild (Bradshaw et al. 1998; Bradshaw &

Schemske 2003; Lexer et al. 2004; Martin et al. 2006;

Noor & Feder 2006). Thus, plants offer attractive study

This journal is q 2008 The Royal Society

3024 C. Lexer & A. Widmer Review. Genic view of plant speciation

objects for addressing fundamental questions inspeciation genetics. The purpose of this review is todiscuss recent progress of the field and the questionsthat are emerging from this work. We avoid discussingthe nature of plant species as this topic has beencovered recently (Rieseberg et al. 2006). Also, we referto Rieseberg & Willis (2007) for an overview on currentprogress in plant speciation and to Ramsey & Schemske(1998), Soltis et al. (2004) and Comai (2005) forpolyploid speciation in particular (see also Hegartyet al. (2008) in the present issue). Here, we focus onrecent speciation genetic work in selected groups ofdiploid plants (diploid in terms of current meioticbehaviour rather than evolutionary history) in whichrapid progress has been made in the last few years.A focus on diploids allows us to discuss questions ofgeneral interest to botanists and zoologists, which maycontribute to much-needed discussion between thesegroups of researchers.

Only few potential plant speciation genes, genesinvolved in the evolution or maintenance of speciesbarriers, have been characterized at the molecular level.These include: hybrid sterility genes conferring cyto-plasmic male sterility (CMS), that is, the loss ofcoordination between nuclear and organellargenes inherited from different parents (Chase 2007);myb-type transcription factors as major determinants offlower colour-linked pollinator shifts (Hoballah et al.2007); and genes involved in hybrid necrosis, i.e. auto-immune responses presumably mediated by rapidlyevolving pathogen-resistant genes (Bomblies & Weigel2007). Fortunately, studies of genic species barrierscan also commence without knowledge of the actualgenes involved. It is important to point this out, as thisfact was not expressed clearly in Wu’s (2001a) land-mark paper: the relative roles of selection and drift indriving speciation may be assessed using the analyticaltoolkits of quantitative evolutionary genetics andpopulation genomics (Lynch & Walsh 1998; Luikartet al. 2003; Beaumont 2005) even before speciationgenes have been identified. A more important pre-requisite is knowledge of the nature and type of barriersamong the taxa studied. As long as there is someevidence that barriers are primarily genic rather thanchromosomal (e.g. evidence from cytogenetics orcomparative genetic mapping), plant evolutionarygeneticists can begin to study the architecture ofgenic species isolation.

Studies of Arabidopsis thaliana and its relatives havecontributed greatly to our understanding of plantevolutionary genetics, and much of this work has beenreviewed recently (Mitchell-Olds 2001; Mitchell-Olds &Schmitt 2006; Lysak & Lexer 2006; Bomblies & Weigel2007). Here, we review recent work of relevance in fiveother plant genera that have become or are currentlyemerging as model systems for plant evolutionarygenetics: Mimulus (monkey flowers); Iris (irises);Helianthus (sunflowers); Silene (campions); and Populus(poplars, aspens, cottonwoods; table 1). Specifically, welook for answers to the following questions of currentinterest: (i) how often do genes of major effect contributeto plant speciation, (ii) do the architectures of genicspecies barriers differ between pairs of species of thesame plant group, (iii) what are the relative roles of

Phil. Trans. R. Soc. B (2008)

endogenous (intrinsic) versus exogenous (environ-mental) genic barriers in particular cases, (iv) are speciesdifferences in general, and trait differences involved inreproductive isolation in particular, more likely to arisevia selection or drift, (v) under which conditions can thebreakdown of reproductive barriers result in adaptiveintrogression, (vi) what are the roles of pleiotropy orlinkage in facilitating or constraining speciation, (vii)how do interspecific barriers relate to and interact withbarriers to gene flow within species, and (viii) whichexperimental approaches may be used to study speciesbarriers in situ in their ecological context? Table 1 listswhich of these eight questions were addressed by eachreviewed study. We mine the literature for answers andclose with an outlook on future research.

2. GENETIC ARCHITECTURE OF REPRODUCTIVEBARRIERS AND HABITAT ADAPTATIONIN MimulusThe genus Mimulus is highly diverse both in terms ofspecies numbers and in adaptations to ecologicalfactors ranging from diverse pollinators and matingsystems to extreme habitats. Members of the genushave been the subject of intensive ecological andevolutionary genetic research for over 50 years andhave become a model system for studies on the geneticsof reproductive isolation and speciation (Wu et al.2008; see also Lowry et al. 2008).

A detailed study on the different components ofreproductive isolation between two Mimulus species,Mimulus lewisii and Mimulus cardinalis, found that mostreproductive isolation between these two taxa occursprior to hybrid formation (Ramsey et al. 2003; Martin &Willis 2007). A central component of such prematingreproductive isolation in animal-pollinated angio-sperms are thought to be specific pollinators (see alsoCozzolino & Scopece 2008; Lowry et al. 2008).Analyses of the genetic architecture of floral traitsdifferentiating Mimulus species that attract differentpollinators consistently found evidence for a role ofmajor-effect genes in the control of phenotypic traitdifferences (Bradshaw et al. 1995, 1998). This impliesthat either individual genes of individually large effector linked clusters of genes with a large cumulative effectcan play a central role in the evolution of plant reproduc-tive isolation and speciation (Bradshaw et al. 1998).

The evidence for a role of major-effect genes inreproductive isolation gained from quantitative traitlocus (QTL) analyses was confirmed in experimentalanalyses of pollinator preference. For example, analysisof pollinator visitation in a highly polymorphic F2

population derived from a cross between M. lewisii andM. cardinalis found that an allele that increased petalcarotenoid concentration reduced bee visitation by80%, whereas an allele that increased nectar pro-duction doubled hummingbird visitation (Schemske &Bradshaw 1999). A follow-up analysis of the adaptivevalue of alternative alleles at the former locus, referredto as YELLOW UPPER5-7 ( YUP) locus, using near-isogenic lines (NILs) in field experiments revealed thatboth YUP alleles in the foreign background increasedvisitation of the alternative pollinator approximately70-fold. These experimental pollinator analyses

Tab

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(Continued

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Review. Genic view of plant speciation C. Lexer & A. Widmer 3025

Phil. Trans. R. Soc. B (2008)

Table

1.(Continued.)

stu

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3026 C. Lexer & A. Widmer Review. Genic view of plant speciation

Phil. Trans. R. Soc. B (2008)

provide strong support for the notion that major-effect

genes are involved in premating reproductive isolation(Bradshaw & Schemske 2003).

In contrast to the genetic architecture of floral trait

differences involved in pollinator attraction, QTLanalyses of traits involved in interspecific mating system

differences between inbreeding and outbreedingMimulus species indicate that these trait differences

(floral morphology and stigma–anther separation) areprimarily controlled by loci with small effect (Lin &

Ritland 1997; Fishman et al. 2002). An analysis of

intraspecific divergence between annual and perennialMimulus guttatus came to the same conclusion (Hall

et al. 2006). A comparison of the genetic architecturecontrolling floral trait divergence, both within divergent

populations of M. guttatus and between M. guttatus and

Mimulus nasutus (Fishman et al. 2002), implied thatthere may be a shared genetic basis for floral divergence

within and among species of Mimulus (Hall et al. 2006).An important aspect is that these analyses of floral

trait divergence repeatedly found strong linkage amongfloral traits (Lin & Ritland 1997; Fishman et al. 2002).

Furthermore, inferred QTL often affected more than

one trait, thus contributing to the genetic correlationbetween those traits (Lin & Ritland 1997; Fishman

et al. 2002). An intraspecific analysis in M. guttatusrevealed high degrees of pleiotropy as well (Hall et al.2006), thus indicating a probable role for genetic

correlations in both inter- and intraspecific barriers togene flow between these Mimulus taxa.

Reproductive isolation among Mimulus species is notonly brought about by floral trait divergence associated

with different pollinators or shifts in mating system, butalso by postzygotic intrinsic barriers that may have

contributed to species isolation at a later stage. The first

direct genetic analyses of hybrid incompatibilities inMimulus were performed by Macnair & Christie

(Macnair & Christie 1983; Christie & Macnair 1984;see recent review of this work by Wu et al. 2008).

Notably, these studies not only provided evidence for the

existence of Dobzhansky–Muller incompatibilities inthis species, but also further established a clear link

between reproductive isolation and habitat adaptation.F1 lethality was observed in crosses between copper

tolerant and non-tolerant populations of M. guttatus,with a gene responsible for copper tolerance in adapted

populations pleiotropically affecting reproductive

isolation between tolerant and non-tolerant populations.This suggests that habitat adaptation may lead to

reproductive isolation and ultimately to speciationthrough interactions with intrinsic isolating factors.

More recently, Fishman & Willis (2001) analysed

patterns of sterility in F1 and F2 hybrids betweenM. guttatus and M. nasutus and found evidence for

Dobzhansky–Muller incompatibilities contributing tothe sterility between the two species. Analysis of marker

segregation in the F2 generation further revealed that

nearly half of the markers exhibited significanttransmission ratio distortion, and allowed identification

of 11 transmission ratio distorting loci (TRDL)throughout the genome. Negative interactions bet-

ween the heterospecific genomes are the most likelycause of the observed pattern, which is in line with

Review. Genic view of plant speciation C. Lexer & A. Widmer 3027

Dobzhansky–Muller incompatibilities existing betweenthe two species (Fishman et al. 2001).

Further loci involved in heterospecific incompat-ibility between these two Mimulus species wereidentified upon examination of male-sterile F2 hybrids.Nearly complete hybrid male sterility was found toresult from a simple genetic incompatibility between asingle pair of heterospecific loci in the investigatedMimulus species pair (Sweigart et al. 2006). Interest-ingly, the alleles causing genetic incompatibility werefound not to be fixed within species. Instead, one of theloci, hms1, was polymorphic in the outcrossingM. guttatus and only locally distributed, which limitsits potential to consistently contribute to the barrierbetween sympatric populations of the two species(Sweigart et al. 2007).

In addition to negative interactions among hetero-specific nuclear loci, Fishman & Willis (2006) foundthat cytonuclear incompatibility occurs in someM. guttatus!M. nasutus hybrids that carry a particularM. guttatus cytoplasm. Hybrids have a pollenless antherphenotype that is caused by a cryptic CMS factor. Inhermaphroditic M. guttatus populations carrying theCMS cytoplasm, the necessary nuclear restorer ispresent, but interspecific hybrids lack the co-evolvedrestorer locus and express the male-sterile phenotype(Fishman & Willis 2006). Case & Willis (2008) foundthat hybrid male sterility in Mimulus is associated with aspecific mitochondrial rearrangement and that theCMS cytotype is highly restricted geographically inM. guttatus and is absent in M. nasutus (Case & Willis2008). These results support the idea that selfishcytoplasmic elements may play an important role inshaping patterns of plant hybrid incompatibilities(Levin 2003).

3. GENETIC ANALYSIS OF SPECIESBOUNDARIES AND ADAPTIVEINTROGRESSION IN LOUISIANA IRISESThe Louisiana irises form a classical example ofintrogressive hybridization in plants (Anderson 1949;Arnold 1997). Early molecular marker studies sup-ported the long-standing hypothesis that introgressivehybridization has played an important role in theevolution of the three ‘model’ irises Iris fulva, Irishexagona and Iris brevicaulis (reviewed by Arnold et al.2004). It has been argued that habitat disturbance mayplay a role in the ecology and evolution of the Louisianaarises. This may include both natural disturbance dueto temporary flooding in the drainage areas where thesespecies occur and man-made disturbance in morerecent times (Arnold et al. 2004). Here, we focus onrecent genetic mapping studies of species boundariesand adaptive introgression in I. fulva and I. brevicaulis.

Initial progress in genetic map-based studies ofspecies boundaries in I. fulva and I. brevicaulis washampered by the size and complexity of their genomes,but this obstacle was recently overcome by usingretrotransposon display markers to map speciesboundaries (Bouck et al. 2005). A surprising resultof this work was that only a relatively small proportion ofmarkers (15%) showed transmission ratio distortions inthe interspecific backcross populations compared with

Phil. Trans. R. Soc. B (2008)

other interspecific mapping studies in plants (Boucket al. 2005). Also, most of these distortions were due toan over- rather than an under-representation ofintrogressed alleles, which suggested that introgressedalleles may often have a positive fitness effect in their newgenetic background (Bouck et al. 2005). Nevertheless,the authors cautioned that the over-representation ofheterospecific alleles may also stem in part frominbreeding depression caused by the experimentaldesign used for mapping. Also, considering the frequentoccurrence of transposable elements in Iris spp. (Boucket al. 2005), it is possible that ‘selfish’ transposableelement activity contributed to the observed trans-mission ratio distortions as well, in analogy to the selfishelement argument discussed for Mimulus above.

In a QTL study of post-establishment mortalityinvolving reciprocal backcrosses between I. fulva andI. brevicaulis in a controlled greenhouse environment,only a small proportion of the genome contributed tohybrid survivorship (Martin et al. 2005). For eight outof nine candidate genomic regions associated withsurvivorship in the two backcrosses, introgressed allelesincreased survivorship. This provided additional sup-port for the idea that introgression in Louisiana irisesmay sometimes contribute to the evolutionarydynamics of naturally occurring hybrid zones (Martinet al. 2005). Nevertheless, the fitness of introgressantsunder natural conditions depends on several additionalisolating mechanisms described below.

A transplantation study with reciprocal backcrossesof the same two species under highly selective fieldconditions provided a first glimpse of the geneticarchitecture of differences in survivorship associatedwith habitat divergence in the wild (wet versus dryconditions; Martin et al. 2006). While field survivor-ship in the backcross to I. fulva was influenced greatlyby negative epistasis between two QTL, introgressedI. fulva alleles tended to increase survivorship in thebackcross towards I. brevicaulis. The latter findingsuggests that traits responsible for adaptation to wetI. fulva habitat can be transferred into I. brevicaulis viaintrogressive hybridization (Martin et al. 2006).

Genetic mapping in interspecific crosses of I. fulvaand I. brevicaulis was also used to explore the geneticarchitecture of flowering phenology, floral traits andpollinator preference in this group (Bouck et al. 2007;Martin et al. 2007, 2008). Although in both cases theauthors cautioned that sample sizes were limited,heritable variation detectable as QTL was identifiedfor all three suites of traits (Bouck et al. 2007; Martinet al. 2007). In the case of flowering phenology, theresults were consistent with selection rather than driftpromoting divergence in flowering time in the evolution-ary history of these two species. This was concludedsince both additive and epistatic effects of QTL allelesgenerally were in the direction expected from theirparental species origin. In the case of morphologicalfloral traits, frequent co-localization of QTL on thegenetic map provided a straightforward explanation forwhy natural late-generation Iris hybrids often exhibitparental-like phenotypes: QTL for five of the nine traitsexamined were co-localized on a single linkagegroup, which implies a high potential for joint trans-mission of these traits in hybrids (Bouck et al. 2007).

3028 C. Lexer & A. Widmer Review. Genic view of plant speciation

Genetic mapping of QTL affecting pollinator (hum-mingbird, lepidopteran and bee) preferences indicatedthat pollinator behaviour poses a much weaker barrier togene flow in these species compared with floweringphenology. Nevertheless, co-localization of floral andpreference QTL also showed that pollinators dorespond to heritable floral traits (Martin et al. 2008).

Clearly, ongoing work in Louisiana irises is begin-ning to reveal which genetic factors are most criticalto reproductive isolation between I. fulva andI. brevicaulis. Flowering time loci and QTL for floraltraits acting as prezygotic barriers (Bouck et al. 2007;Martin et al. 2007) and QTL associated with habitatdivergence as a potential pre- or postzygotic barrier(Martin et al. 2006) appear to be more important interms of barrier strength than intrinsic isolating factors(Bouck et al. 2005). It will be interesting to compare thegenetic architectures and genomic distributions of allthese different types of isolating factors once this seriesof mapping experiments is complete (see Martin et al.2008), and to compare the sizes of selection coefficientsassociated with different types of barriers. If adaptiveintrogression is important in irises, then we may expectthat the fitness benefits of introgressed alleles in certainenvironments outweigh the reduction in fitness due topostzygotic intrinsic factors. Similarly, we would expectthat genetic correlations (linkage or pleiotropy) alongthe chromosomes are of a type that facilitates ratherthan constrains adaptive introgression.

4. THE GENETIC ARCHITECTURE OF BARRIERSTO INTER- AND INTRASPECIFIC GENE FLOWIN HelianthusStudies of Helianthus have contributed greatly to ourunderstanding of the molecular genetic architecture ofbarriers to gene flow in outcrossing plants. Here, wefocus on the studies that inform us about the geneticbasis of barriers to introgression between diploidHelianthus species, and largely ignore the increasingbody of data on hybrid or ‘recombinational’ speciationin this group (e.g. see Rieseberg et al. 2003).

Among early molecular marker studies of introgres-sion in Helianthus (Arias & Rieseberg 1994; Kim &Rieseberg 1999; Rieseberg et al. 1999a), the last one isof particular relevance as it was among the first to useQTL mapping to address the potential of adaptive traitintrogression in plants. Kim & Rieseberg (1999) usedQTL mapping to study the genetic architecture ofintrogression from the wild sunflower speciesHelianthus debilis into subspecies texanus of Helianthusannuus. The results revealed a simple architecture ofgenetic isolation—only 11 out of 60 QTL controllingmorphological species differences were linked to pollensterility factors or otherwise negatively selectedchromosomal blocks. For the remaining 45 QTL nobarrier to introgression was detected, and introgressionof just three small linkage blocks appeared sufficient torecover the phenotype of H. annuus ssp. texanus. Thissupported the hypothesis that subspecies texanus ofH. annuus originated through introgression, althoughthe authors cautioned that data on the adaptive value ofcritical QTL are required to ultimately test thishypothesis (Kim & Rieseberg 1999). We note that

Phil. Trans. R. Soc. B (2008)

some of the phenotypic traits under selection inH. annuus ssp. texanus have been identified morerecently (Whitney et al. 2006).

In addition to QTL studies of experimental crosses,genomic studies of natural interspecific hybrid zonesproved highly informative for studying the architectureof barriers to gene flow in Helianthus (Rieseberg et al.1999b; Gardner et al. 2000; Buerkle & Rieseberg2001). Rieseberg et al. (1999a,b) used a genome-widepanel of dominant molecular markers to study patternsof introgression across three ‘replicate’ hybrid zones ofH. annuus and Helianthus petiolaris. Out of 26 genomicsegments with significantly reduced introgression inthese hybrid zones, 16 were associated with pollensterility, and only 50% of the barrier to introgressionwas due to chromosomal rearrangements segregatingbetween the two species. This indicated that the speciesbarrier had a complex basis that included bothchromosomal and genic isolating factors (Rieseberget al. 1999b). A characteristic feature of these earlystudies of Helianthus hybrid zones was that increased orreduced introgression often was observed over largecentimorgan distances or even over entire linkagegroups. This is expected based on the availableknowledge of linkage disequilibrium (LD) in hybridzones (Barton & Hewitt 1985), since LD close to thecentre of a hybrid zone will effectively increase the sizeof chromosomal blocks affected by either positive ornegative selection (Rieseberg & Buerkle 2002; Yatabeet al. 2007).

A recent study of the same two sunflower speciesbased on more than 100 codominant microsatellite lociand 14 sequenced genes revealed a more refinedpicture of the genetics of species isolation in thisgroup (Yatabe et al. 2007). In that study, geneticdivergence for individual marker loci between popu-lations of the two sympatric congeners H. annuus andH. petiolaris was much weaker than that in theprevious studies of hybrid zones (Rieseberg et al.1999b; Buerkle & Rieseberg 2001), even after correct-ing for differences in variability between loci. The onlyhighly divergent ‘outlier’ loci were five microsatellitesassociated with expressed sequence tags (ESTs), andthese five loci were put forward as candidate locipotentially under divergent selection (Beaumont 2005;Yatabe et al. 2007). For most loci, divergence betweenH. annuus and H. petiolaris was also much weaker thanbetween either of these two species and the closelyrelated allopatric Helianthus argophyllus (Yatabe et al.2007). Furthermore, increased divergence nearchromosome breakpoints extended only as far as5 cM. These results suggest that even strong andgenetically complex isolating barriers are unlikely toprevent widespread introgression.

It is important to note that the population genomicwork by Yatabe et al. (2007) was based on contrasts ofpopulations that lacked early generation hybrids, asopposed to the hybrid zone studies outlined earlier. LDin populations of these self-incompatible outcrosserswill be much lower than that in hybrid zones(Rieseberg & Buerkle 2001; Flint-Garcia et al. 2003).Theory predicts that the effect of linkage on genedispersal will decrease rapidly over generations, asneutral or advantageous alleles are recombined into a

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Figure 1. QTL analysis in a complex interspecific pedigreefacilitates the simultaneous mapping of inter- and intraspe-cific QTL potentially involved in ecological divergence inHelianthus. Linkage group 4 of Helianthus is shown as anexample. Marker positions are indicated by horizontal barsalong the linkage group. Shown as blocks to the right of thegroup are interspecific QTL positions and support limits withinterspecific effect sizes indicated by the horizontal width ofeach block. All interspecific QTL positions were also testedfor the presence of intraspecific QTL, and positions at whichintraspecific QTL polymorphism was found are shown inblack. The average effect size of intraspecific QTL (notshown) was less than one-third that of interspecific QTL.STEMDIA, stem diameter; RGR, relative growth rate;MG, magnesium uptake; LFAREA, leaf area; LFWDTH,leaf width; SEEDWT, seed weight. Modified from Lexeret al. (2005a).

Review. Genic view of plant speciation C. Lexer & A. Widmer 3029

foreign genetic background (Barton & Hewitt 1985;Martinsen et al. 2001). At this stage, ‘hitch-hiking’ willbe confined to short genomic distances and the fate ofintrogressed alleles will depend mainly on their ownfitness effects (Martinsen et al. 2001; Yatabe et al.2007), as also predicted by the genic view of the processof speciation (Wu 2001a).

Studies of barriers to gene flow in Helianthusincreasingly aim at addressing both barriers betweenand within species. A large QTL study on the geneticsof species differences in sunflowers facilitated thesimultaneous mapping of interspecific morphological,physiological and life-history QTL segregating betweenH. annuus and H. petiolaris, and intraspecific QTLsegregating between the different H. petiolaris backcrossparents used to form the mapping cross (figure 1; Lexeret al. 2005a). A comparison of QTL effect sizesindicated that intraspecific QTL generally had smallereffects than interspecific QTL, consistent with theprediction that larger QTL are more likely to spread tofixation across a subdivided species (Rieseberg & Burke2001; Morjan & Rieseberg 2004). By contrast, smallerQTL may be trapped in local populations and are thusmore likely to be visible as allelic substitutions at thewithin-species level (figure 1; Lexer et al. 2005a).

Studies of selective sweeps and local adaptationwithin wild sunflower species are informative in thiscontext as well (Kane & Rieseberg 2007). This studyused 128 EST-derived microsatellite loci to reveal thegeographical extent of selective sweeps in H. annuus

Phil. Trans. R. Soc. B (2008)

sampled from typical habitat of this species in the plainsof central/western North America and from distinctarid desert and brackish salt marsh habitats. Spatiallyseparated populations often diverged at some loci whileevolving in concert at other loci, and selective sweepsassociated with local adaptation (e.g. salt tolerance)were detected at three different geographical scales(Kane & Rieseberg 2007). Studies such as this cancontribute to our understanding of both cohesion ofsets of populations within species and the origin ofbarriers between species.

5. ECOTYPE FORMATION, HYBRIDIZATIONAND INTROGRESSION IN SileneThe genus Silene encompasses approximately 700species that display great morphological diversity andhave colonized a wide range of habitats, ranging fromthe sea coast to heavy metal-contaminated soils, andhigh alpine tundra vegetation. The genus includesspecies that are either hermaphroditic, gynodioecious(hermaphrodites and females coexist) or dioecious(males and females coexist; Desfeux et al. 1996).

A common and widespread European herb with agynodioecious mating system is Silene vulgaris, thebladder campion. It is well known for its ability tocolonize heavy metal-rich soils, such as mines orserpentine soils. Bratteler and co-workers have inves-tigated the genetic architecture of morphological andphysiological traits that differentiate two S. vulgarisecotypes, one that grows on serpentine soil (naturallyheavy metal-rich soil), and one on non-serpentine soil.A genetic linkage map based on amplified fragmentlength polymorphism markers (Bratteler et al. 2006b)provided the foundation for the identification ofQTL for morphological and physiological traitspotentially involved in serpentine adaptation (Bratteleret al. 2006a,c). The observed genetic architecturesuggests that traits potentially involved in habitatadaptation are controlled by few genes of major effectand have evolved under divergent selection (Bratteleret al. 2006c).

The studies on S. vulgaris ecotypes also revealedstrong phenotypic correlations between traits, andQTL underlying this variation displayed extensivelinkage. Clustering of QTL for different traits can bedue to either pleiotropy or linkage and both can eitherconstrain or facilitate adaptation, depending onwhether trait correlations have positive or negativefitness effects (Bratteler et al. 2006c). Based on theobserved combination of QTL directions, it wassuggested that further ecotype divergence may begenetically constrained and may thus slow down orprevent further divergence between ecotypes.

Silene vulgaris is related to a clade of dioecious Silenespecies, of which the white campion, Silene latifoliaPoiret (syn. Melandrium album Garcke, syn. Melandriumpratense Roehl.), is arguably the best known. The sexof individual plants is genetically determined bysex chromosomes. Males are heterogametic (XY) andfemales are homogametic (XX; Vyskot & Hobza 2004).Silene latifolia and the closely related Silene dioicarepresent an interesting model to study the process ofinterspecific differentiation at the genome level because

3030 C. Lexer & A. Widmer Review. Genic view of plant speciation

they are able to hybridize when they come into contact,yet maintain morphological integrity at the boundariesof such hybrid zones (Minder et al. 2007). The twospecies differ most prominently in floral traits andhabitat preferences (Karrenberg & Favre 2008). Hybridzones between the two species were found to bedominated by late-generation backcross hybrids,whereas intermediate, presumably early generationhybrids, are rare. This indicates that gene flow is likelyto occur between the two species, with the hybrids actingas bridges to gene flow (Minder et al. 2007).A population genomic analysis of differentiation andidentification of outlier loci between S. latifolia andS. dioica revealed that the species boundary has beenshaped by a combination of introgression and selection(Minder & Widmer 2008). Introgression occurs as aconsequence of hybridization upon secondary contact,but species differences appear to be maintained outsideof hybrid zones, presumably as a consequence ofselection acting on phenotypic traits involved inpollinator or habitat adaptation. These results challengethe idea of whole-genome isolation (Mayr 2001; Wu2001a), and support the idea that genomes can beporous and that species differentiation has a genic basis(Minder & Widmer 2008).

6. Populus AS A ‘MODEL TREE’ FOR STUDYINGTHE ECOLOGICAL AND EVOLUTIONARYGENOMICS OF SPECIES ISOLATIONTwo parallel developments call for the inclusion of thewind-pollinated, primarily dioecious tree genus Populusin this review: (i) it is among the few tree genera withthree-generation pedigrees available, and theseresources are increasingly being used to map speciesisolation factors and barriers to introgression (Frewenet al. 2000; Yin et al. 2004) and (ii) closely relatedspecies of Populus are currently being used to adopt theconcepts of ‘admixture mapping’ (Chakraborty &Weiss 1988) from human medical genetics to plantspeciation genetics, and the first results of this work arenow becoming available (Lexer et al. 2007). A numberof additional factors render Populus an attractive modelfor speciation genetics: ecologically divergent speciesoften co-occur in sympatry or parapatry and areinterfertile (Eckenwalder 1996); widespread syntenysuggests that species barriers are genic rather thanchromosomal (Cervera et al. 2001); and a completegenomic sequence, genetic maps and many thousandsof ESTs are available (Cervera et al. 2001; Yin et al.2004; Sjodin et al. 2006; Tuskan et al. 2006). As a resultof these developments, the speciation genetic literatureon Populus is growing rapidly, paralleled by only a fewother tree genera (e.g. oaks; Muir et al. 2000;Scotti-Saintagne et al. 2004).

Genetic linkage and QTL mapping were usedsuccessfully to map traits involved in interspecificdifferentiation between the two North American speciesPopulus deltoides and Populus trichocarpa, with a QTLanalysis of bud set/bud flush being perhaps the mostrelevant example with respect to ecological differen-tiation (Frewen et al. 2000). Two candidate genesinvolved in photoperiod were found to co-localize withbud set and bud flush QTL, thus providing a rare

Phil. Trans. R. Soc. B (2008)

example for the molecular characterization of ecologicalQTL in trees. The much larger linkage mapping studyby Yin et al. (2004) between the same two speciesrevealed large-scale heterospecific segregation distor-tion. Large contiguous blocks of donor genome on twolinkage groups were distorted in a manner consistentwith positive selection, thus suggesting a role for geneticcorrelations in the maintenance of species barriers inPopulus in sympatry or parapatry (Yin et al. 2004). Someof the genetic correlations involved in barriers amongPopulus species may be the result of reduced recombina-tion in specific regions of the genome carryingecologically relevant genes, such as the peritelomericregion of chromosome XIX of Populus. This region hasbeen shown to carry a large number of NBS–LRR(nucleotide-binding site/leucine-rich repeat)-type resist-ance genes and it also harbours several features expectedfor an incipient sex chromosome, such as a genderdetermination locus, distorted segregation and highlevels of haplotype divergence (Yin et al. 2008).

The second approach, admixture mapping (associ-ation mapping in recombinant admixed populations),has been highly successful in human medicalgenetics (Reich et al. 2005; Zhu et al. 2005) but isonly beginning to be applied to evolutionary biology.In Populus, first indications for the usefulness of theapproach came from genetic studies of the NorthAmerican cottonwoods Populus fremontii and P. trichocarpa(Keim et al. 1989; Martinsen et al. 2001). A molecularmarker study of a natural hybrid zone between thesespecies indicated great inter-locus variation in thegeographical extent of introgression (Martinsen et al.2001). Genomic analyses of hybrid zones are currentlytaken further in the ecologically divergent hybridizingEuropean species Populus alba (white poplar) andPopulus tremula (European aspen; figure 2; Lexeret al. 2005b, 2007). Hybrid zones of these two speciescontain a high proportion of recombinant backcrosses(Lexer et al. 2005b), and admixture LD decays quicklywith successive backcross generations (Lexer et al.2007). Although both clonality and assortative matingappear to contribute to fine-scale spatial patterns inhybrid zones of P. alba and P. tremula, these potentialsources of LD can be accounted for by widening thespatial level of sampling (van Loo et al. 2008). Thus,interspecific Populus hybrid zones can serve as naturallaboratories for the identification of candidate lociinvolved in species isolation (Lexer et al. 2007; Joseph &Lexer 2008). An important notion is that in Populus,candidate gene loci can be subjected to rigorous tests ofgene function, because the necessary tools for thistype of functional genomics work are available (Brunneret al. 2004; Sjodin et al. 2006; Tuskan et al. 2006).

7. SYNTHESIS AND EMERGING QUESTIONSFOR FUTURE RESEARCHThe genic view of the process of speciation can providea framework for rigorous tests of the mechanisticunderpinnings of and evolutionary forces operatingduring speciation (Rieseberg & Burke 2001; Coyne &Orr 2004; Noor & Feder 2006). Studies with a focus onspeciation and differential adaptation were consideredjointly in this review, since adaptation may result in

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Figure 2. Introgression of two codominant DNA-basedmarkers (a, O220; b, O149) across a Central Europeanhybrid zone of P. alba and P. tremula, relative to neutralexpectations. X-axes: molecular hybrid index as an estimateof average genome-wide introgression. Y-axes: genotypefrequencies for each locus. The introgression of thepredominant P. tremula allelic class (black solid lines,homozygotes; black dashed lines, heterozygotes) fromindividuals with low hybrid index (P. tremula) to those withhigh hybrid index (P. alba) varies among loci. Grey regionsindicate the 95% confidence envelope for logistic regressionsfit to simulated data under the assumption of neutralintrogression for each locus (dark grey region, homozygotes;light grey region, heterozygotes). The slope of the regressionfor homozygotes but not for heterozygotes departs signi-ficantly from neutrality for the marker in (a). Modified fromLexer et al. (2007).

Review. Genic view of plant speciation C. Lexer & A. Widmer 3031

speciation if there is pleiotropy or linkage between loci

involved in reproductive isolation and loci under

divergent selection (Felsenstein 1981; Via 2001).Note that, in principle, adaptation may be sufficient

to achieve isolation if extrinsic postzygotic isolation isstrong (e.g. late-generation hybrids are outperformed

by F1 genotypes in hybrid zones among Rhododendronspecies in the absence of strong intrinsic postzygotic

barriers, Milne et al. 2003).

In plants, most research programmes currently usegenetic mapping of TRDL and QTL and population

genomic scans to study the genetic architecture ofporous genomes (table 1). The reviewed studies may

not represent a random draw of species from nature,but they may inspire related work on different groups of

Phil. Trans. R. Soc. B (2008)

taxa so that generalizations can be drawn with ever-increasing precision. We suspect that new develop-ments in molecular systematics (e.g. DNA-barcoding;Chase et al. 2005) will increasingly facilitate both theidentification of species pairs for in-depth genetic studyand the interpretation of speciation genetic data in acomparative framework.

Our present review of recent progress with a focus onfive plant genera suggests that the genetic architecture ofspecies isolation differs greatly between taxa. Never-theless, a direct involvement of selection in speciation (asopposed to divergence due to drift alone) can no longerbe ignored, and future work can now aim at under-standing functional aspects and ecological impacts of theselected traits and genes. A ‘genic view of speciation’ isparticularly useful for this purpose, because the explicitconsideration of recombination and selection (‘locus-specific effects’) potentially allows evolutionarybiologists to disentangle the different mechanismsoperating during divergence. The following topicsemerge as being of special interest for future researchon the nature of genic barriers to gene flow in plants.

(a) The role of intraspecific variation

in speciation

Intraspecific variation is an often neglected aspect inplant speciation genetics. Only three of the reviewedstudies addressed it explicitly, namely the genomicanalysis of replicate hybrid zones in Helianthus byBuerkle & Rieseberg (2001), the simultaneous inter-and intraspecific QTL analyses of ecological speciesdifferences in Helianthus by Lexer et al. (2005a,b;figure 1), and the genetic analysis of natural variationfor hybrid incompatibility in Mimulus by Sweigart et al.(2007). More speciation genetic studies are needed thatexplicitly address this topic. This is important to(i) understand the fate of incompatibility alleles withinspecies and ask whether such alleles evolve in an adaptiveor neutral fashion at the within-species level (Sweigartet al. 2007), (ii) learn whether intraspecific variation forspecies isolation is due to polymorphism of isolationfactors or rather due to differences in exogenous(ecological) selection pressures in different parts of aspecies’ range (Buerkle & Rieseberg 2001), (iii) addressthe potential of evolution from standing variation versusevolution from new mutations (Hermission & Pennings2005) and (iv) improve our understanding of theinteractions between species differentiation and speciescohesion (Ehrlich & Raven 1969; Morjan & Rieseberg2004). Species cohesion and species differentiationcannot be viewed in isolation, as both will depend oninteractions of the same evolutionary forces, such asgene flow, selection and drift (Ehrlich & Raven 1996;Morjan & Rieseberg 2004). Thus, speciation geneticstudies should increasingly integrate analysis of inter-and intraspecific components of variation.

(b) Balancing versus directional selection

in population genomic scans

An increasing number of speciation genetic studiesuse genome scans for species differentiation to charac-terize porous genomes (Scotti-Saintagne et al. 2004;Savolainen et al. 2006; Yatabe et al. 2007; Minder &Widmer 2008). A shortcoming of most existing genome

3032 C. Lexer & A. Widmer Review. Genic view of plant speciation

scans is that they often fail to identify loci underbalancing selection, i.e. outlier loci in the lower tail ofthe distribution of divergence estimates (Beaumont &Balding 2004). This is the case in part because thespecies pairs tested are typically sympatric and have ahistory of frequent gene exchange, thus the lower 95%confidence limit of divergence (FST or GST) will bebiased towards zero (Scotti-Saintagne et al. 2004;Savolainen et al. 2006; Yatabe et al. 2007; Butlin et al.2008), which makes it difficult to distinguish betweenneutral loci and loci under balancing selection (Beau-mont & Balding 2004). Consequently, neutral expec-tations used to identify outliers in the upper tail may bebiased. A rare exception is the study of thewell-differentiated hybridizing Silene species,S. latifolia and S. dioica (Minder & Widmer 2008);that study was able to identify loci under divergent andloci under balancing selection.

One question of great interest to students of porousgenomes is whether most of the genome is permeable togene flow with occasional low-recombination ‘hotspotsof differentiation’, or whether most of the genome isprotected from gene flow except for occasional ‘pores’(Mayr 2001; Wu 2001a,b). Answering this questionwith population genomic scans will require reliableneutral expectations for outlier detection. As a start,more studies are needed that scan pairs of populationsand species with different degrees of relatedness andgeographical overlap to ‘calibrate’ the outlier testscurrently in use.

(c) The timing of fixation of major- versus

minor-effect alleles

The order of fixation of ‘major’- and ‘minor’-effectalleles that differ in their effect on the phenotype duringadaptation has sometimes been considered to be clear.Theory predicts an ‘adaptive walk’ that starts with largesteps that become smaller as a population nears afitness optimum (Orr 1998). Recent theoretical work,however, indicates that minor-effect alleles may befixed before major ones when the selection pressureincreases gradually over time or, in other words, ifenvironmental change is slow (Collins et al. 2007;Kopp & Hermisson 2007). In this context, themeaningful estimation of effect sizes (Zmagnitudes)of QTL involved in adaptation and speciation is ofmajor interest.

As shown by Lexer et al. (2005a,b) in the case ofHelianthus and Bouck et al. (2007) for Iris, themagnitudes of QTL effects sometimes differ greatlywhen estimated as ‘per cent variance explained’ (PVE)in the mapping population, relative to the interspecificphenotypic gap or relative to levels of standingvariation. To effectively address the size distributionand timing of factors fixed during adaptation andspeciation, QTL studies should try to estimate andinterpret the magnitudes of QTL effects in all threeways in parallel whenever possible; for example, theestimates of standing variation and interspecific gapscould be obtained from population samples used tocharacterize quantitative traits prior to genetic map-ping. Consistently small interspecific QTL, as observedfor floral traits involved in mating system divergence inMimulus (Fishman et al. 2002), may then indicate an

Phil. Trans. R. Soc. B (2008)

important role for the preferential fixation of minor-effect QTL. This may be the case if the adaptive walk islimited by the rate of environmental change (Kopp &Hermisson 2007); for example, the selective environ-ment in which mating system divergence occurred inMimulus may have changed gradually. This wouldmake sense—mating system divergence (the transitionfrom outcrossing to selfing or vice versa) is known to beaffected by complex fitness trade-offs (Fishman et al.2002) and mixed mating is common in plants(Lande & Schemske 1985). On the contrary, some ofthe floral traits differentiating Iris species with differentpollination syndromes were of large effect regardlesswhether they were measured in terms of PVE, standingvariation or the interspecific gap (Bouck et al. 2007).This lends additional support to saltational changesassociated with abrupt shifts in pollination syndromes,as also observed in previous landmark studies ofpollinator isolation (Bradshaw et al. 1995; Schemske &Bradshaw 1999; Bradshaw & Schemske 2003). We notethat common criteria for the definition of major- andminor-effect QTL are available only for effect size interms of PVE, but not in terms of standing variationor species difference. Clearly, more empirical andtheoretical work is needed to assess the likelihood offixation of QTL measured on these scales.

(d) The likelihood of adaptive introgression

Several of the reviewed studies touched on thelikelihood of adaptive introgression in plants, the QTLanalyses of species boundaries in Iris (Martin et al. 2005,2006) and population genomic studies in Helianthus(Kane & Rieseberg 2007; Yatabe et al. 2007) beingexcellent examples. The Helianthus studies clearly showthat even complex barriers to introgression are per-meable to gene flow (Yatabe et al. 2007) and thatbeneficial alleles at some loci will spread across largedistances in outcrossing plants (Kane & Rieseberg2007), thus contributing to species cohesion (Morjan &Rieseberg 2004). An open question is whether such‘cohesion genes’ often introgress across speciesboundaries due to their adaptive value, or whetherthey are more likely to be contained within species.

Owing to the paucity of genetic studies addressingadaptive introgression, it is too early to say for surewhich plant traits are likely to favour this process.Nevertheless, judging from molecular marker data inIris (reviewed by Arnold et al. 2004) and Populus (vanLoo et al. 2008), mixed sexual/asexual reproductivesystems appear to favour introgression, possibly byfacilitating the persistence of ecologically successfulgenotypes in the face of meiotic difficulties. Populationgenomic scans and admixture mapping in naturalhybrid zones hold great promise for studying adaptiveintrogression in situ. This is facilitated by new approa-ches of detecting locus-specific effects in hybrid zones(figure 2), which allow the detection of both negativeand positive departures from neutral introgression(Lexer et al. 2007).

(e) Speciation genes

Identifying the molecular identity and function of genesinvolved in species barriers in plants will remain animportant task for evolutionary geneticists, and the

Review. Genic view of plant speciation C. Lexer & A. Widmer 3033

methodologies required to achieve this goal are

available (reviews by Feder & Mitchell-Olds (2003)

and Noor & Feder (2006)). The QTL and population

genomic studies reviewed here represent a starting

point for the molecular characterization of loci involved

in species isolation. Experimental transplants or natural

hybrid zones should allow the estimation of selection

coefficients of these genes under realistic conditions.

Studies of incipient species or divergent ecotypes will be

relevant as they allow the detection of genes directly

involved in reproductive isolation, as opposed to

isolation factors that evolved post-speciation. It will be

interesting to see which functional classes of genes most

often contribute to genic barriers in plants, which types

of regulatory networks are involved and how these genes

interact with other types of isolation factors, e.g.

chromosomal rearrangements (Navarro & Barton

2003). These questions can be addressed using the

tools of ecological and evolutionary functional genomics

(Feder & Mitchell-Olds 2003; Noor & Feder 2006),

which are becoming available for several of the plant

groups reviewed here. We look forward to seeing the

results of this research over the coming years.

We thank Vincent Savolainen, Salvatore Cozzolino, NolandMartin, Douglas Schemske, Jay Sobel and two anonymousreferees for their helpful comments on the manuscript. C.L.’swork on within-species variation for genomic isolation inEuropean Populus is supported by grant NE/E016731/1 of theBritish NERC (Natural Environment Research Council) andA.W.’s work on Silene is funded by SNF grants 3100AO-104114 and 3100A0-116455.

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